Re: [ccp4bb] crashing-out protein eluted from Nickel column

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: indexing multiple lattices
From: "Winter, G (Graeme)" g {- dot -} winter {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-02-18

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From: Clemens Vonrhein vonrhein {- at -} GLOBALPHASING {- dot -} COM
Date: 2008-02-18

Subject: Re: crashing-out protein eluted from Nickel column
From: "Nadir T {- dot -} Mrabet" Nadir {- dot -} Mrabet {- at -} MEDECINE {- dot -} UHP-NANCY {- dot -} FR
Date: 2008-02-18

Hi,

One has to bear in mind that adsorption at high pH (above 6.5 is already
high for Imac; pKa of exposed His is 6.2) leads to stronger interactions
which, in turn, require stronger eluting conditions, e.g. > 200 mM
imidazole (pKa ~ 7.0).
I suggest lowering the pH of the adsortion buffer, e.g. 6.5 or even 6.0
(try and see) so that you dont need to go beyond 100 mM
imidazole in your elution buffer (as mentioned earlier, you must ajust
the pH of the latter as required with extra HCl).

Best,

Nadir Mrabet

--

Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet@medecine.uhp-nancy.fr

Ronnie Berntsson wrote:
> Hi,
>
> In addition to the tips already suggested, you could also try to elute
> your protein with histidine.. I've got a protein that crashes out with
> imidazole as well, and I have succesfully used 200mM histidine for
> elution (using either KPi or MES buffer in my case).
>
> Cheers,
> Ronnie
>
> On Feb 15, 2008, at 5:47 PM, Juliana Barbosa Coitinho wrote:
>
>>
>> Dear all,
>> I am with the same problem of Jacob.
>> My protein is precipitating, especially when I 'freeze it'. I am
>> using a His Trap HP column coupled with a desalting column. Already
>> tried to elute with gradient of imidazole (that allowed an elution
>> with less amount of imidazole), but even then, the protein still
>> crashes-out.
>> I am trying to use the protein immediately after purified it, but
>> this is not always possible and I am losing much protein with that.
>> I will try to use the tips that have already been said.
>> But if someone can help me more, I will thank!!!
>>
>> Thanks a lot!
>>
>> Juliana
>>
>> ------------------------------------------------------------------------
>> Abra sua conta no Yahoo! Mail
>> ,
>> o único sem limite de espaço para armazenamento!
>
>
>
> Ronnie Berntsson
>
> ----------
> Ph.D. Student
> Department of Biochemistry
> Groningen Biomolecular Sciences and Biotechnology Institute
> & Zernike Institute for Advanced Materials
> University of Groningen
> Nijenborgh 4, 9747 AG
> Groningen, The Netherlands
>
> telephone: +31 50 363 4195
> telefax: +31 50 363 4165
> e-mail: r.berntsson@rug.nl
> homepage: http://www.rug.nl/gbb/research/researchgroups/enzymology/index
>
>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: indexing multiple lattices
From: "Winter, G (Graeme)" g {- dot -} winter {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-02-18

Next message:
Subject: Re: indexing multiple lattices
From: Clemens Vonrhein vonrhein {- at -} GLOBALPHASING {- dot -} COM
Date: 2008-02-18